Genetic structure of the European polecat (Mustela putorius) and its implication for conservation strategies C. Pertoldi1,2,3, P. Breyne4, M. T. Cabria5, D. Halfmaerten4, H. A. H. Jansman6, K. Van Den Berge4, A. B. Madsen3 & V. Loeschcke1 1 Department of Ecology and Genetics, University of Aarhus, Ny Munkegade, Aarhus C, Denmark 2 Department of Applied Biology, Estaci ´on Biol ´ ogica Do˜ nana, CSIC, Pabell ´on del Peru´ , Seville, Spain 3 Department of Wildlife Ecology and Biodiversity, National Environmental Research Institute, Rønde, Denmark 4 Institute for Forestry and Game Management, Gaverstraat, Geraardsbergen, Belgium 5 Departamento Zoologia y BCA, Facultad Farmacia, Universidad del Pais Vasco, Vitoria-Gasteiz, Spain 6 Alterra-Centre for Ecosystem Studies, AA Wageningen, The Netherlands Keywords polecat; Mustela putorius; microsatellite DNA; population structure; assignment test; bottleneck; population fragmentation; postglacial recolonization. Correspondence Cino Pertoldi, Department of Ecology and Genetics, University of Aarhus, Building 540, Ny Munkegade, DK-8000 Aarhus C, Denmark. Fax: +4586127191 Email: [email protected] Received 20 July 2005; accepted 23 November 2005 doi:10.1111/j.1469-7998.2006.00095.x Abstract During the last century, the European polecat Mustela putorius populations in most of Europe declined and survived in fragmented patches, because of habitat alterations and direct persecution. To assess the genetic consequences of the demographic decline and to describe the spatial pattern of genetic diversity, 250 polecats sampled at seven localities from five European countries – Poland, Denmark (southern Denmark and northern Denmark), Spain, Belgium (eastern and western) and the Netherlands – were screened by means of nine microsatellite loci. Genetic diversity estimated by mean expected heterozygosity (HE) and allelic richness (AR) were moderately high within populations [range: 0.50 (northern Denmark) ≤ HE ≤ 0.64 (Poland) and 1.33 ≤ AR ≤ 7.80] as compared with other carnivores and mustelids. Bottleneck tests suggested that polecat populations in southern Denmark and Poland have declined recently and populations from northern Denmark and the Netherlands have expanded recently, whereas the remaining populations did not show any sign of demographic change. Recent demographic changes could suggest that some of the populations are still not in equilibrium, which could partly explain the relatively high genetic variability observed in polecat populations despite the drastic decline in population size observed in several European countries. A significant heterozygote deficiency [FIS =0.19; 0.01 ≤ 95% confidence interval (CI) ≤ 0.32] suggests substructuring within the total European sample. Partitioning of the genetic variation among sampling locations (FST =0.14; 0.06 ≤ 95% CI ≤ 0.23) and pairwise FST between localities (range: 0.01 ≤ FST ≤ 0.37) without any correlation with the geographic distances between localities were found, suggesting a recent divergence and a restriction of gene flow between populations and the action of genetic drift. An assignment test showed that the Polish and the northern Danish populations were the most unique, whereas the other populations were partially admixed. Factorial component analysis tests indicate a subdivision of the total sample into two distinct groups: one including the samples from Poland and the two Danish localities and the second group comprising the remaining localities investigated. The observed pattern of genetic differentiation is suggested to be due to two main routes of recolonization after the last glacial period. To compare the results obtained with microsatellite data, the most variable region of the mitochondrial DNA (d-loop) was sequenced and different phylogenetic reconstructions and genetic diversity analyses based on nucleotide (π) and haplotype diversity (h) measures within populations were performed using a subsample of populations. The lack of well-defined geographical structure, as well as the reduced level of mitochondrial DNA variability (p: 0.00274 ± 0.00038; h: 0.876 ± 0.028) that was found, has been previously reported in several studies on different carnivores and supports the hypothesis of post-glacial recolonization from southern or eastern refugees of Europe as suggested by the microsatellite data. Implications for conservation strategies of the polecat at the European level are discussed. Introduction Present status of the polecat in Europe The polecat Mustela putorius is a medium-sized carnivore that lives in many parts of Europe (Mitchell-Jones et al., 1999). Its preferred habitat is near bodies of water, like freshwater lakes, rivers and wetlands. Polecats are also found on the edges of forests and grasslands with islands of scrub trees. The polecat is listed in Appendix III of the Bern Convention (European Council, 1979, http://www.eko.org.pl/1kp/prawo_html/konw_bernenska_z3.html) and in Appendix V of the European Community Habitats & Species Directive (1992, http://europa.edu.int/comm/environment/nature/nature_conservation/eu_nature_legislation/habita ts_directive/index_en.htm). The polecat is of some conservation significance as it is considered to be vulnerable in most parts of Europe and threatened in some parts (Birks & Kitchener, 1999). Its distribution range and population densities have decreased in several European countries over the past few decades (Santos Reis, 1983; Vigna Taglianti, 1988; Weber, 1988; Blanco & Gonzalez, 1992; Saint-Girons et al., 1993; Stubbe & Stubbe, 1994; Libois, 1996; Baghli & Verhagen, 2003; Møller et al., 2004). In some other countries however, as for example the Netherlands or in some regions of France, the status of the polecat has been reported as stable (Hollander & Van der Reest, 1994). In Flanders, the northern part of Belgium, polecats never lost the status of ‘commonly spread’ although densities started to decline some decades ago (Van Den Berge & De Pauw, 2003). In Denmark, according to the Danish game bag record the number of polecats killed by hunting during the last 60 years has been declining (Møller et al., 2004). Habitat fragmentation and degradation and more particularly the drainage of wetlands as well as changes in the agricultural landscape have been suggested as the principal causes for the decrease of populations of the polecat in Europe (Blanco & Gonzalez, 1992). Because this mustelid is supposed to be affected by habitat fragmentation and degradation (Bright, 1993), it is considered as an indicator species in human impact studies. Genetic and demographic consequences of habitat fragmentation In conservation biology there is a growing awareness of the consequences of habitat loss and over-exploitation, which often leads to a reduction in population sizes. Today, habitat fragmentation is considered as one of the most serious threats to the survival of animal populations. This is a result of the spatial structuring of populations caused by the loss of area, the reduction in area of remaining fragments and increased distance between fragments (Gerlach & Musolf, 2000). Habitat fragmentation produces a reduction in genetic diversity because of a loss of variability due to low effective population size (NE) and an increased genetic differentiation between remaining fragments. Population subdivision is therefore expected to reduce the adaptive potential that a population has to face up to environment change (Lacy, 1997; Bijlsma, Bundgaard & Boerema, 2000a). Declining adaptive variation might interact negatively with the demographic consequences of habitat loss, raising the risks of extinction (Bijlsma et al., 2000a). The effect of fitness reduction observed in populations with low NE is thought to be partly the result of increased homozygosity for (partially) recessive deleterious alleles (Charlesworth & Charlesworth, 1987). Several studies have documented inbreeding depression in wild populations (reviewed in Keller & Waller, 2002). The substantial variation in the level of inbreeding depression between populations and their consequences seem to be strongly taxon dependent (Crnokrak & Roff, 1999; Keller & Waller, 2002). Therefore, there is a need for a concerted effort to study patterns of genetic variation in a wide variety of taxa at different spatial scales. Studies of spatial population structure in mammals have revealed that most mammalian populations are genetically subdivided, with relatively small NE and low dispersal rates that are consequences of the social and mating systems. The scale of subdivision varies considerably, suggesting that genetic structure is influenced by complex interactions between social organization, dispersal tendencies and environmental factors (Chepko-Sade & Halpin, 1987). We wanted to assess the likelihood for persistence of the polecat populations studied to estimate the genetic variability within the populations and to describe the patterns of genetic differentiation among the populations. Recent reduction of NE can generate large genetic distances among populations and may drastically reduce the genetic variability within populations in a short time period (Hedrick, 1999). Molecular data based on microsatellite variation were chosen for our investigation as microsatellites are highly informative and are currently used in many studies addressing conservation issues (Kyle, Davis & Strobeck, 2000; Kyle, Robitaille & Strobeck, 2001; Walker et al., 2001; Kyle, Davison & Strobeck, 2003). Due to their high mutation rate (fast evolving), microsatellites